Novel and unusual genes for nitrogen and metal cycling in Planctomycetota- and KSB1-affiliated metagenome-assembled genomes reconstructed from a marine subsea tunnel

Abstract The Oslofjord subsea road tunnel is a unique environment in which the typically anoxic marine deep subsurface is exposed to oxygen. Concrete biodeterioration and steel corrosion in the tunnel have been linked to the growth of iron- and manganese-oxidizing biofilms in areas of saline water seepage. Surprisingly, previous 16S rRNA gene surveys of biofilm samples revealed microbial communities dominated by sequences affiliated with nitrogen-cycling microorganisms. This study aimed to identify microbial genomes with metabolic potential for novel nitrogen- and metal-cycling reactions, representing biofilm microorganisms that could link these cycles and play a role in concrete biodeterioration. We reconstructed 33 abundant, novel metagenome-assembled genomes (MAGs) affiliated with the phylum Planctomycetota and the candidate phylum KSB1. We identified novel and unusual genes and gene clusters in these MAGs related to anaerobic ammonium oxidation, nitrite oxidation, and other nitrogen-cycling reactions. Additionally, 26 of 33 MAGs also had the potential for iron, manganese, and arsenite cycling, suggesting that bacteria represented by these genomes might couple these reactions. Our results expand the diversity of microorganisms putatively involved in nitrogen and metal cycling, and contribute to our understanding of potential biofilm impacts on built infrastructure.


Introduction
The marine deep biosphere comprises a significant part of life on Earth (Bar-On et al. 2018 ), but it is still lar gel y unexplor ed.The Oslofjord subsea tunnel in Norway is a unique environment in which the marine deep subsurface, typically comprised of anoxic sediments and jointed r oc k mass, is exposed to oxygen in the tunnel.This subsea road tunnel has a maximum depth of 134 m below sea le v el and is cov er ed by sprayed concrete, employed dir ectl y onto the r oc k mass, r einforced with steel fibers for r oc k support of the tunnel structure.Ho w ever, cracks in the bedrock allow seepage of saline water from the overlying water column through the bedrock and across the sprayed concrete layer.In areas of the tunnel with water seepage, a biofilm has de v eloped on the spr ayed concr ete surface, causing biodeterior ation of the concrete with associated steel fiber corrosion (Kara či ć et al. 2018 ).The biofilm consists of an outer orange to bro wn lay er , rich in amorphous iron hydroxide (ferrihydrite), and an inner black layer, rich in manganese oxide biominerals (Na-buserite , todorokite , and birnessite) (Hagelia 2007(Hagelia , 2011 ) ). Reduction of these ir on hydr oxides, manganese oxides and, additionally, sulfate, has been detected in some biofilms (Hagelia 2011, Kara či ć et al. 2018 ).
Biotic and abiotic reactions within the biofilm lead to acidification of the saline water from pH 7.5-8 to 5.5-6.5 at low water flow rates (Hagelia 2011 ).A likely responsible mechanism for the acidification is microbial oxidation of Fe 2 + and Mn 2 + with oxygen, whic h, upon pr ecipitation of Fe 3 + and Mn 4 + biominer als, r eleases H + (Manahan 2000 ).Ho w e v er, these r eactions can also occur at circumneutral pH (Emerson 2000 ).Additionally, the penetration of chloride and the deposition of Mn-oxides is known to cause pitting corrosion on steel (Dickinson et al. 1997, Olesen et al. 2001, Hagelia 2011 ).The acidic water causes deep disintegration and enhances the porosity of the cement paste matrix due to dissolution of portlandite and calcium silicate hydrate, leading to formation of carbonates, thaumasite sulfate attack and magnesium attack (Ha gelia 2011, Kar a či ć et al. 2018 ).
Based on these pr e vious studies, metal-cycling micr oor ganisms were expected to be abundant in biofilms.Howe v er, when the 16S rRNA gene diversity of biofilm samples collected from three tunnel areas was analyzed (Kara či ć et al. 2018 ), microbial communities were surprisingly dominated by putative nitrogen-cycling members: the most abundant amplicon sequence variant (ASV) across 64 biofilm samples was affiliated with the ammonium-oxidizing archaeon Nitrosopumilus .Other highly abundant ASVs were affiliated with betaproteobacterial ammonium-oxidizing Nitrosomonadaceae , marine nitrite-oxidizing Nitrospina , nitrifying Nitrospira , and marine anaerobic ammonium-oxidizing (anammox) Candidatus Scalindua (Kara či ć et al. 2018 ).Additionally, a followup metagenomics study identified in these biofilms a novel family of anammox bacteria named Ca.Anammoxibacteraceae (Suarez et al. 2022 ).T hese results suggested that no v el micr oor ganisms enriched in Oslofjord tunnel biofilms could perform metabolic reactions linking nitrogen and metal biogeochemical cycling.
Her e, we r econstructed meta genome-assembled genomes (MAGs) from Oslofjord tunnel biofilm samples r epr esenting abundant community members affiliated with no vel taxa.T his study aimed to identify the metabolic potential for novel nitrogen-and metal-cycling reactions, thus expanding the known diversity of micr oor ganisms with the potential of linking these cycles .T his resulted in the selection of 33 MAGs affiliated with the phylum Planctomycetota and candidate phylum KSB1, which were interrogated with respect to their potential biogeochemical repertoire.Typicall y, both phyla hav e br oad metabolic potential and ar e implicated in heter otr ophic lifestyles.Planctom ycetota ar e fr equentl y described as extr emel y div erse bacteria with unusual cell biology and aerobic or facultative anaerobic, chemoheterotrophic metabolism (Elshahed et al. 2007, Spring et al. 2018, Wiegand et al. 2018 ), with the exception of the anaerobic lithoautotrophic anammox bacteria (Kartal et al. 2012 ).Similarl y, while no r epr esentatives of the candidate phylum KSB1 have been cultured to date, MAG analyses indicate that these microorganisms are likely involved in organic carbon degradation and fermentation in estuarine (Baker et al. 2015 ) and hydrothermal sediments (Dombrowski et al. 2017 ), harboring genes encoding multiple carbohydrateactive enzymes (López-Mondéjar et al. 2022 ) and potentially novel isopr opanol dehydr ogenases (Dalcin Martins et al. 2019 ).
In particular, we searched for both canonical and divergent marker genes involved in nitrogen cycling pathwa ys .T hese included anaerobic ammonium oxidation via a reductive hydroxylamine oxidoreductase-encoding gene ( hao ) for nitrite reduction to nitric oxide (Ferousi et al. 2021 ), hydrazine synthase ( hzsABC ) for ammonium oxidation coupled to nitric oxide r eduction, pr oducing hydrazine (Dietl et al. 2015a ), and hydrazine dehydrogenase ( hdh ), for hydrazine oxidation to dinitrogen gas (Maalcke et al. 2016 ).A gene encoding hydro xylamine o xidase ( hox ), with unknown physiological function but conserved in anammox bacteria (Kartal and Keltjens 2016 ), was included in our analyses.We also searched for genes in aerobic (complete) nitrification (van Kessel et al. 2015 ) via ammonium monooxygenase ( amoABC ), for ammonium oxidation to h ydroxylamine, h ydr oxylamine oxidor eductase ( hao ), for hydroxylamine oxidation to nitrite, and nitrite oxidoreductase ( nxrABC ) for nitrite oxidation to nitrate (Daims et al. 2016a ).Genes in the denitrification pathway (Philippot 2002 ) comprised both membrane-bound ( narGHI ) and periplasmic ( na-pAB ) nitr ate r eductases for nitr ate conv ersion to nitrite, nitrite r eductase for nitrite reduction to nitric oxide ( nirK and nirS ) or to ammonium ( nrfAH ), nitric oxide reductase for nitric oxide conversion to nitrous oxide ( norB ), and nitrous oxide reductase for the last step in denitrification, nitrous oxide reduction to dinitrogen gas ( nosZ ).
Ther efor e, in this study, MAGs with potential for iron reduction could also r epr esent micr oor ganisms ca pable of r educing manganese, and ther efor e ar e r eferr ed to as pr esenting gener al metalcycling potential.
MAGs were annotated with DRAM v1.0 (Shaffer et al. 2020 ) with default options, except -min_contig_size 1000, and most genes of interest were searched in annotation files.Additionally, some genes were identified via complementary methods: genes encoding pr oteins involv ed in anammox metabolism wer e searc hed both via annotation files and via blastp analyses using previously identified r efer ence sequences fr om Ca.Kuenenia stuttgartiensis (de Almeida et al. 2016 , Kartal andKeltjens 2016 ), and iron cyclingrelated genes were detected with FeGenie (Garber et al. 2020 ).Phylogenetic tr ees wer e built with FastTr ee v2.1.10( Price et al. 2010 ) and visualized in iToL v6 (Letunic and Bork 2021 ), with the exception of the tree containing UBA1845 MAGs from this study and r efer ence genomes, whic h was built with IQ-TREE v2.2.0 (Minh et al. 2020 ) from an alignment of 74 single copy genes done with GT oT ree v1.7.00 (Lee 2019 ).Heat maps were generated in RStudio v4.2.1 using the v egan pac ka ge v2.6-4 (Oksanen et al. 2019 ).Gene clusters were identified and visualized in R with the standard gggenomes w orkflo w ( https:// github.com/thackl/ gggenomes ).Div er gent sequence similarity anal yses wer e performed with HHpred ( https:// toolkit.tuebingen.mpg.de/tools/ hhpred ).All figures were edited in Adobe Illustrator.

Results and discussion
Planctomycetota -and KBS1-affiliated MAGs were abundant across biofilm samples.
We analyzed our MAG dataset (NCBI BioProject PRJNA755678) for metabolic potential regarding novel nitrogen-and metal-cycling reactions.Upon MAG inspection for accuracy of assembly and binning, 33 MAGs were selected for this study, of which 24 had high quality ( > 90% completeness and < 5% contamination) and 9 had medium quality (here, > 75% completeness and < 8% contamination) (Bo w ers et al. 2017 ).Individually, the MAGs selected for this study r eac hed up to 2.5% of r elativ e abundance in the biofilm community, summing 1.7%-7.6% of the community across biofilm samples (Fig. 1 ), which were collected in four instances between 2016 and 2020 from two tunnel areas: the pump site, with spr ayed concr ete since 1999 for permanent r oc k support, and the test site , with spra yed concrete since 2010 to test concr ete dur ability (Hagelia 2011 ).The retrieved MAGs could not be easily classified beyond the phylum le v el: all four of the candidate phylum KSB1-affiliated MAGs belonged to the putative family 'CR04bin15'.Furthermor e, onl y 6 of 29 MAGs within the phylum Planctomycetota could be classified beyond the putative family level (Supplementary Table 1).Next, based on taxonomic novelty, we focused on searching for genes involved in nitrogen and metal cycling.
Genes with sequence similarity to hydrazine synthase subunits were present in several phycisphaerae MA Gs.
Anaerobic ammonium oxidation (anammox) is an important process in the nitrogen cycle and is catalyzed by the enzyme hydrazine synthase, encoded by three genes ( hzsABC ) used as markers for this metabolism (Harhangi et al. 2012 ).We identified 21 genes that had blastp hits with a bitscore > 40 to hzsABC from Ca. Kuenenia stuttgartiensis across 17 genomes in this study (Supplemental Table 1), hereafter referred to as hzs -like genes.While a minim um bitscor e v alue of 60 is the default used for DRAM annotations (Shaffer et al. 2020 ), we used this low bitscore threshold to allow for the identification of div er gent sequences.
Se v er al important genes potentially implicated in anammox metabolism were detected in seven MAGs affiliated with the class Phycisphaerae , within the putative family UBA1845: OFTM5,174,250,285,286,321,and 371 (Figs. 2 and 3 ).These included 10 hzsABC -like genes with blastp-derived bitscore values ranging from 89 to 163 (in the annotation range) against hzsABC from Ca. Kuenenia stuttgartiensis (Fig. 2 ), as well as similar values when hzsABC sequences from Ca. Scalindua or Ca.Anammoxibacter were used.In these Phycisphaerae MAGs, hzsB -and hzsClike genes were fused, as it has been observed in marine anammox Ca.Scalindua species (van de Vossenberg et al. 2013a, Dietl et al. 2015b ), and had an hzsA -like gene encoded immediatel y upstr eam (Supplementary Table 1, Fig. 2 ).Similarl y, we found hzsABC -like genes in three reference genomes (GCA_016 208 685.1, GCA_020 344 555.1 and GCA_022 563 615.1) affiliated with Phycisphaerae UBA1845, with hzsA immediately upstream of fused hzsBC -lik e subunits (Fig. 2 ).Ad ditionally, we identified in these MAGs genes annotated as hydroxylamine oxidoreductases ( hao and, only in OFTM5, also hox ), nitrate/nitrite oxidoreductases ( narGHI or nxrABC ), R/b complex genes, ETM subunit 1 and 2encoding genes, and other nitrogen cycle-related genes (Fig. 3 for a summary and Supplementary Table 1 for each gene annotation in each MAG).Ho w ever, no h ydrazine deh ydrogenase-or nitrite reductase-encoding genes ( hdh , nirK , or nirS ) were identified in any genomes from this study.Furthermore, genes encoding subunits of oxygen reductases were detected in five of these se v en MAGs, and genes encoding a nitric oxide reductase, periplasmic nitrate reductase , manganese , and iron oxidases were prevalent in Phycisphaerae genomes (Fig. 3 ).Analyses of reference genomes related to Phycisphaerae UBA1845 MAGs in our study indicated that these micr oor ganisms ar e pr esent in marine sediments and gr oundwater, as well as in w astew ater and drinking water treatment plants (Fig. 4 ).
Based on these results, we hypothesize that these se v en Phycisphaerae MAGs within the family UBA 1845 could represent novel anammox bacteria outside the order ' Ca.Br ocadiales', whic h holds all curr entl y described and hypothesized anammox taxa (Kartal et al. 2012, Suarez et al. 2022, Zhao et al. 2022 ), requiring future experimental validation by enrichment cultures and 15 N isotope studies .T he missing hydrazine dehydrogenaseencoding gene of the new MAGs could be too divergent to be detected based on sequence similarity or, alternativ el y, the identified hydro xylamine o xidoreductase could be involved in hydrazine oxidation to dinitrogen gas, an activity previously shown in vitro in Ca.Kuenenia stuttgartiensis (Maalcke et al. 2016 ), relying on a cross-link ed acti ve site heme (REF).Oxygen reductase genes present in these genomes might support the function of oxygen tolerance or detoxification, which has been recently described in anammox bacteria in bioreactors (Yang et al. 2022 ) and aquifer ecosystems (Mosley et al. 2022 ).Furthermore, MAGs comprising a nov el clade II gr oup of Ca.Br ocadiae, likel y anammox bacteria, wer e r econstructed fr om oxygenated aquifer samples and also lac ked a hydr azine dehydr ogenase-encoding gene (Mosley et al. 2022 ), as in our study .Finally , nitr ate-dependent ir on oxidation has been reported in Ca.Brocadia and Ca.Scalindua enrichment cultures (Oshiki et al. 2013 ), and metal oxide respiration has been described in Ca.Kuenenia stuttgartiensis, Ca.Brocadia, and Ca.Scalindua species (van de Vossenberg et al. 2013b, Strous et al. 2006, Oshiki et al. 2016 ), supporting the potential for metal-cycling metabolism detected in these Phycisphaerae MAGs that could repr esent nov el anammox bacteria.Other MAGs in this study were not considered to represent potentially novel anammox because hzsABC -like genes in these MAGs had a low bitscore value (40-60) from blastp analyses using Ca.K. stuttgartiensis r efer ence sequences, hzsA was not immediately upstream or downstream of hzsBC , and few anammox metabolism genes were identified in these genomes.
No vel nitr a te/nitrite oxidoreductase genes w ere present in planctomycetota -affiliated genomes.
Figure 2. Phylogenetic tree of hzsB and hzsC (-like) genes (concatenated protein sequences unless indicated as fused genes.Bold indicates reference sequences r etrie v ed fr om NCBI with r espectiv e accession numbers, while the other sequences wer e obtained fr om this study.Onl y sequences with an hzsA gene located upstream of hzsBC were included in the tree.Bitscore values were obtained from blastp hits (Supplementary Table 1) to Ca. Kuenenia stuttgartiensis HzsB and HzsC sequences, r espectiv el y, pr esent in the tree .T he tree was rooted in the Brocadiales (upper) clade.and the methane o xidizer Ca.Methylomirabilis o xyfera.All of these genes were part of NarGHI/NxrABC clusters in our MAGs, indicating that they likely encode novel nitrate/nitrite oxidoreductases.
While we could not assign a reaction direction (nitrite oxidation or nitrate reduction) based on our sequence analyses, we hypothesize that sequences in the first cluster (orange in Fig. 5 ) could r epr esent NxrA, giv en the pr e v alence of nitrite oxidizers in this cluster and the widespread presence of genes encoding oxygen r eductases, hydr ogenases, and formate dehydr ogenases in the 19 MAGs in this cluster (Fig. 3 and Supplementary Table 1).On the other hand, we hypothesize that 18 sequences in the second cluster (green in Fig. 5 ) could r epr esent NarG, giv en the pr e v alence of nitr ate r educers in this cluster.We hypoth- esize that these putativ e nitr ate r educers could hav e a r ole in the observed steel fiber corrosion in the tunnel, as the activity of nitr ate-r educing bacteria has been pr e viousl y linked to metal corr osion, potentiall y via extr acellular electr on tr ansfer (Miller et al. 2018, Iino et al. 2021 ).Out of 19 Planctomycetota MAGs with putativ e nov el Nrx-type nitrite oxidor eductase-encoding genes, six MAGs ( Planctomycetota OFTM77, Planctomycetota PLA2 OFTM341, Pirellulaceae OFTM348, Planctomycetaceae OFTM22, Bythopirellula OFTM389, and Bythopirellula OFTM39) also had putative Nar-type nitr ate r eductase-encoding genes (Fig. 3 and 5 ), similar to the Chloroflexota -affiliated nitrite oxidizer Ca.Nitr ocalder a r obusta, which harbors two types of Nar/Nxr (Spieck et al. 2020 ).
Most putative nxr -harboring MAGs had low-and/or highaffinity oxygen reductase genes and, frequently, norB, napAB , and hao (Fig. 3 and Supplementary Table 1).We infer that these MAGs could r epr esent putativ el y nov el nitrite oxidizers with metabolic versatility to oxidize alternativ e substr ates coupled to a variety of terminal electron acceptors (oxygen, nitrate, nitric oxide, and ferric ir on).Giv en that pr e viousl y described nitrite oxidizers affiliate to the phyla Proteobacteria , Chloroflexota , Nitrospirota , and Ni-trospinota (Daims et al. 2016b ), this is the first report of putative nitrite oxidation potential in the phylum Planctomycetota .Genes encoding manganese, arsenite or iron oxidases were present in 12 of the 19 MAGs with putativ e nov el nxr genes, indicating potential for metabolic versatility related to metal(loid) oxidation in these organisms (Fig. 3 ).Such potential agrees with versatility in substr ate oxidation pr e viousl y r e ported for nitrite o xidizers of the genus Nitrospira (Koch et al. 2015, Bayer et al. 2021 ) and expands the potential for metabolic versatility in putative nitrite oxidizers.

Clusters of genes encoding proteins likely involved in nitrogen cycling were conserved across genomes.
We identified a conserved gene cluster together with putative nitrogen c ycling-inv olved proteins across several genomes (Fig. 6 ).In 13 instances (Supplementary Table 1), putative Nar-encoding genes wer e pr esent upstr eam of a six-gene cluster encoding (1) a multi-heme c -type cytochrome (MHC) with, most frequently, five heme-binding motifs (5MHC in Fig. 6 ), (2) a 4Fe-4S dicluster domain-containing pr otein fr equentl y fused to a mol ybdopterin oxidoreductase (mbd in Fig. 6 ), (3) a polysulphide reductase NrfD-type putative membrane subunit, (4) an alternative complex III tr ansmembr ane subunit actD , (5) a cbb 3 -type cytoc hr ome c oxidase tr ansmembr ane subunit ccoP , and (6) a transmembr ane quinol:cytoc hr ome c oxidor eductase quinone-binding subunit 2 ( ccoII ).This gene cluster had the arc hitectur e of an iontr anslocating ener gy-tr ansducing membr ane complex containing an NrfD-like subunit, but did not matc h an y pr e viousl y described complexes (Calisto and Per eir a 2021 ).Ther efor e, based on HHpr ed div er gent sequence similarity anal yses and on the pr esence of upstream putative Nar-encoding genes, we hypothesize that it could r epr esent a nov el membr ane-bound NrfAH-like nitrite r eductase , which con verts nitrite to ammonium.Alternatively, these genes could encode for a protein part of the r espir atory electr on tr ansport c hain, giv en that, in fiv e instances, oxygen r eductase genes were downstream of the gene cluster (Fig. 6 ).Additionally, we identified in two MAGs (OFTM8 and OFTM33) a similar gene cluster, missing the molybdopterin oxidoreductase, ccoP, and ccoII , downstream of Nap-and putative Nxr-encoding genes, and, in one MAG (OFTM248), a similar gene cluster downstream of a porincytoc hr ome c complex for iron reduction (Fig. 6 ).This further suggests a potential role for proteins encoded by this gene cluster in r espir atory electr on tr ansfer.
Potential for high metabolic versatility was detected in MAGs affiliated with the phyla KSB1 and planctomycetota .
We identified a variety of genes encoding pr oteins involv ed in nitrogen, oxygen, sulfur, and metal(loid) cycling in MAGs in this study (Supplementary Table 1), suggesting potential for high metabolic versatility in the microorganisms represented by these MAGs (Fig. 3 ).All four KSB1-affilated MAGs (OFTM72, 153, 177, and 356) had r espir atory potential, with genes encoding nitr ate, oxygen, and ir on r eductases, as well as sulfhydrogenase genes for elemental sulfur reduction to sulfide with dihydrogen gas production (Fig. 3 ).Only one nosZ gene was detected in this study, in OFTM356 (Supplementary Table 1).Additionally, the KSB1 MAGs had genes encoding arsenite and iron o xidases, hydro xylamine o xidoreductase, and genes with low sequence similarity to hzsABC from Ca. Kuenenia stuttgartiensis (Fig. 3 and Supplemental Table 1).
These results provide further evidence for the role of KSB1 bacteria in nitrogen cycling and expand the potential for high metabolic versatility in the KSB1 phylum.A r ecent, compr ehensiv e anal ysis of 44 nonr edundant, high-quality KSB1 MAGs r econstructed fr om gr oundwater, bior eactors, and marine ecosystems pr e viousl y identified metabolic potential for carbohydrate and hydr ocarbon degr adation potentiall y coupled to oxygen and nitr ogen r espir ation ( narG , nrfA , nosZ , and cydAB genes) in KSB1 bacteria (Li et al. 2022 ).Given the low sequence similarity to canonical enzymes and the lack of an operon structure, we infer that hzsABClike genes in our KSB1 MAGs are unlikely to encode a hydrazine synthase.Instead, we hypothesize that the pr e v alence of hzs -like genes with low sequence similarity to canonical anammox genes in MAGs from this study indicates that hydrazine synthase-like enzymes may comprise a br oader, widespr ead enzymatic family with potential for activity with alternative substrates.
While all MAGs in our study had potential for nitrogen cycling, 26 of 33 MAGs also had potential for metal(loid) cycling, suggesting that bacteria r epr esented by these genomes might couple these reactions.Of 29 Planctomycetota MAGs, 15 had genes encoding manganese oxidases, 3 encoding arsenite oxidases, and 5 encoding ir on oxidases, whic h might be coupled to nitrate or oxygen r espir ation in these micr oor ganisms (Fig. 3 ).Additionall y, ir on reduction potential was detected in four Planctomycetota MAGs.A coupling of iron oxidation and nitr ate r eduction has been ob-Figure 5. Midpoint-rooted phylogenetic tree of NarG/NxrA-encoding genes.Reference sequences were retrieved from NCBI and start with accession numbers.Other sequences were obtained from this study and are provided with DRAM annotations as well as bitscore values from blastp hits (Supplementary Table 1) to the Ca.Kuenenia stuttgartiensis NarG/NxrA sequence present in the tree .T he two main clades are color coded in orange and green.serv ed befor e in the family Gallionellaceae (He et al. 2016 ) and the DTB120 candidate phylum (McAllister et al. 2021 ), and this study suggests that it might also occur in Planctomycetota .
To our knowledge, this is the first report of potential for manganese and iron cycling in nonanammox bacteria in the phylum Planctomycetota (Wiegand, Jogler andJogler 2018 , Kappler et al. 2021 ).Ho w e v er, 16S rRNA gene anal yses of microbial mats from an ir on-ric h thermal spring (Selv ar ajan et al. 2018 ), deep sea iron hydro xide de posits (Stor esund and Øvr eås 2013), and metallifer-ous deposits from hydrothermal vents (Storesund et al. 2018 ) have pr e viousl y identified abundant Planctomycetota groups, including Ca. Brocadiales and Phycisphaerae UBA1845.Additionally, the Planctomycetota bacterium Bythoypirellula goksoyri was isolated on organic carbon sources under oxic conditions from deep sea iron hydro xide de posits (Stor esund and Øvr eås 2013 ).In our study, one of three MAGs affiliated with Bythoypirellula had a Cyc2-encoding gene, indicating potential for iron oxidation in these micr oor ganisms, which aligns with their isolation source.These results ex-Figure 6.Genomic regions re presentati ve of common gene clusters potentially encoding novel ion-translocating energy-transducing membrane complexes containing an NrfD-like subunit in MAGs from this study.Genes in the molybdopterin (mbd) oxidoreductase (oxr)-containing gene cluster are color coded in orange and are abbreviated as follows: MHC, multi-heme c -type cytochrome ( cyt c ), with the number of heme-binding motifs indicated ahead; 4F e4S, 4F e-4S dicluster domain-containing protein frequently fused to the molybdopterin oxidoreductase subunit and unless indicated; nrfD, a polysulphide reductase NrfD-type putative membrane subunit; actD , alternative complex III transmembrane subunit D; ccoP , cbb 3 -type cytoc hr ome c oxidase tr ansmembr ane subunit P; ccoII , tr ansmembr ane quinol:cytoc hr ome c oxidor eductase quinone-binding subunit 2; barrel, Cupin domain PF07883.Genes encoding subunits of low-affinity oxygen reductases ( cox ), periplasmic nitrate reductase ( nap ), putative membr ane-bound nitr ate r eductase ( nar ), and putativ e nitrite oxidor eductase ( nxr ) ar e color-coded in pur ple, gr een, blue, and y ello w, r espectiv el y.Some genes of interest upstream or downstream of gene clusters are included: FeTF, Iron-dependent transcriptional regulator; norB , nitric oxide reductase; s70 or s54, regions interacting with these sigma factors; flgS, two-component system sensor kinase of the NtrC family; ctaA , heme a synthase; sco , synthesis of cytoc hr ome c oxidase protein; porin and porin-cytoc hr ome c (PCC) complexes, iron reductases.pand the phylogenetic diversity of microorganisms putatively involved in metal cycling.Together with Zetaproteobacteria , which has been pr e viousl y detected in Oslofjord tunnel biofilms (Kara či ć et al. 2018 ), these bacteria affiliated with Planctomycetota and KSB1 could contribute to iron oxidation in the Oslofjord tunnel, potentially contributing to steel fiber corrosion.Finally, such microorganisms could play a role in microbially-induced corrosion of built infr astructur e in other marine en vironments .

Conclusions
The deep biosphere remains largely unexplored due to sampling costs and challenges.Ho w ever, microbial communities in these ecosystems may harbor untapped potential for novel biogeoc hemical r eactions in the nitr ogen cycle and biotec hnological applications .T his study took adv anta ge of samples fr om a unique, o xygenated dee p marine ecosystem, the Oslofjord tunnel, to explore the potential for such novel metabolic capabilities in micr oor ganisms enric hed in concr ete-degr ading biofilms.We identified potential for nitrogen and metal cycling in novel taxa within the phyla Planctomycetota and KSB1, hypothesizing that these micr oor ganisms might be pr e viousl y unr ecognized anammo x, nitrite-o xidizing, and nitrogen-and metal-cycling bacteria.These results expand the known diversity of microorganisms putativ el y involv ed in these important biogeoc hemical r eactions, and contribute to our understanding of potential biofilm impacts on built infr astructur e.
We thank the following agencies for funding for this research: Dutc h Researc h Council (NWO) SIAM grant 024002002 and Eur opean Researc h Council (ERC) Syner gy gr ant MARIX 854088, aw ar ded to MSMJ, as well as NWO VI.Veni.212.040,aw ar ded to PDM.CS w as supported b y the Adlerbert Resear ch Foundation.Acknowledgements The authors acknowledge support from the National Genomics Infr astructur e in Stoc kholm funded by Science for Life Laboratory, the Knut and Alice Wallenberg Foundation and the Swedish Research Council, and SNIC/Uppsala Multidisciplinary Center for Advanced Computational Science for assis-tance with massiv el y par allel sequencing and access to the UPP-MAX computational infr astructur e. Assembl y and binning were done with resources provided by SNIC through UPPMAX under the projects SNIC 2021-22-112 and2021-23-111.

Figure 1 .
Figure 1.Relative abundance of MAGs in pump station (P) and test site (T) samples collected from the Oslofjord tunnel in four years (2016-2020).Values are provided in Supplementary Table1.

Figure 3 .
Figure 3. Summary of metabolic potential identified in MAGs in this study.MAGs r epr esenting or ganisms with potential for anammox metabolism ar e highlighted in orange, for nitrite oxidation in y ello w, and for other reactions in nitrogen and metal cycling in green.The presence of genes encoding pr oteins involv ed in nitr ogen (N), oxygen (O 2 ), sulfur (S), and metals (iron and manganese) or metalloid (arsenic) c ycling is indicated b y the corresponding metabolic group colors, while the absence of genes is indicated by grey.Proteins are as follows: HzsABC-like, genes with sequence similarity to subunits of hydrazine synthase; Hao, hydroxylamine oxidoreductase; Hcp, hydroxylamine reductase; NarGHI, putative membrane-bound nitr ate r eductase; NxrABC, putativ e membr ane-bound nitrite oxidor eductase; NorB, nitric oxide r eductase; Na pAB, periplasmic nitr ate r eductase; CoxABCD, lo w-affinity c ytoc hr ome c o xidase/o xygen r eductase; CydAB, high-affinity cytoc hr ome bd ubiquinol o xidase/o xygen reductase; Sulfhyd.; sulfhydrogenase/elemental sulfur reductase; MnOx, manganese oxidase; AoxAB; arsenite oxidase; Cyc2, iron oxidase; DFE, Desulfovibrio ferrophilus -like flavin-based extr acellular electr on tr ansfer complex for ir on r eduction; Omc, outer membr ane cytoc hr ome c for ir on r eduction; porin, porin involv ed in ir on r eduction; PCC, porin-cytoc hr ome c complex for ir on r eduction.

Figure 4 .
Figure 4. Biogeogr a phy of Phycisphaerae MAGs affiliated to the family UBA1845.The phylogenetic tree was built using an alignment of 74 single-copy genes (see methods) in MAGs r etrie v ed fr om this study in combination with r efer ence genomes r etrie v ed fr om NCBI, as indicated by accession numbers .T he order Ca .Brocadiales was used as outgroup.Black circles indicate branches with > 95% ultrafast bootstrap support.